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Pisum sativum mutants insensitive to nodulation are also insensitive to invasion in vitro by the mycorrhizal fungus, Gigaspora margarita
plan ience ELSEVIER
Plant Science 102 (1994) 195-203
Pisum sativum mutants insensitive to nodulation are also insensitive to invasion in vitro by the mycorrhizal fungus,
Gigaspora margarita Boovaraghan
B a l a j i *a, A m a d o u
M . B a a'l, T h o m a s Yves Pich6 a
A. L a R u e b, D a v i d T e p f e r c,
aCentre de Recherche en Biologie Forestibre, Facult~ de Foresterie et de G~omatique, Universit~ Laval, Quebec, Quebec, Canada GIK 7P4 bBoyce Thompson Institute for Plant Research, Tower Road, Ithaca, N Y 14853-1801, USA CLaboratoire de Biologie de la Rhizosph~re, lnstitut National de la Recherche Agronomique, Versailles Cedex, F-78026, France Received 8 July 1994; accepted 3 August 1994
Abstract
Transformed root cultures were established using Agrobacterium rhizogenes inoculation of pea mutants, altered in their interaction with Rhizobium. They were tested in an in vitro model for sensitivity to the vesicular-arbuscular mycorrhizal (VAM) fungus, Gigasporamargarita. VAM development was assessed using light and electron microscopy. Two non-nodulating, non-nitrogen fixing (Nod-, Fix-) pea mutants were resistant to VAM colonization in vitro: Mycelium developed on the root surface but failed to colonize the interior. A nodulating (Nod +) genotype, which was unable to fix nitrogen (Fix-) in association with Rhizobium and a parental line, Lincoln (Nod +, Fix+), interacted normally with the fungus, showing extensive internal colonization. These results confirm, under axenic conditions, previous reports showing that defective nodulation is correlated with defective mycorrhization. We propose using this in vitro model to identify factors necessary to initiating and maintaining the VAM/plant symbiosis.
Keywords: VAM; Myc mutants; Pisum sativum; Gigaspora margarita; Agrobacterium rhizogenes; Rhizobium; Genetic transformation
1. Introduction
Vesicular-arbuscular mycorrhizas (VAM) are ubiquitous and associate with most of the vascular * Corresponding author. I Present Address: Institut de Recherche en Biologie et Ecologie Tropicale, BP 7047 Ouagadougou 03, Burkina Faso, West Africa.
plants [1-3], for which they provide essential nutrients and protect againr * drought and plant pathogens. Among 202 species of legumes surveyed, almost 90% engaged in mycorrhizal symbioses, and among these more than 82% were of the VAM type [1]; however, cultures of VAM fungi have not been obtained in the absence of plant host [4], impeding comprehension and manipulation of the symbiosis. The role of the host
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(94)03964-B
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plant, although easier to study, has only recently received attention. Even though there has not been an exhaustive genetic study of competence by legumes to establishing VAM symbiosis, mutants resistant to mycorrhization by the fungus were reported [5,6], and this defect was correlated with defective nodulation by Rhizobium [7]. We performed similar studies using an in vitro model for the VAM/root interaction, in which mutants of Pisum sativum that had been designated either capable (Nod÷) or incapable (Nod-) of establishing VAM symbiosis [7,8] were infected with the VAM fungus, Gigaspora margarita, in the absence of contaminating microorganisms. We thus verified that the Myc- phenotype is correlated with the inability to nodulate (Nod-), but is not correlated with the inability to fix nitrogen (Fix-) in association with Rhizobium. The experimental system made use of transformed root organ cultures, produced by inoculating different pea genotypes with A. rhizogenes. Although normal VAM interactions can take place in axenically grown plants in agar [9-11] or on non-transformed root organ cultures [12], transformed roots grow faster than normal roots [13] and provide a reproducible and simple system for studying VAM symbioses [14-171. This paper reports the isolation of transformed roots from Myc + and Myc- mutants of pea (P. sativum cv. Sparkle) and their interaction with a VAM fungus, Gigaspora margarita, in vitro. Three transformed lines and a parental line, Lincoln, were examined for their response to the mycorrhizal fungus G. margarita.
Agar was included at 15.0 g/l, (Difco Laboratories, Detroit, MI). The same medium without agar (5 ml) was used to culture bacteria for 48 h prior to plant inoculation using a rotary shaker at 100 rev./min at 27°C. Inoculum was prepared by centrifuging bacteria for 10 min at 1775 x g and resuspending them in 20 ml of sterile distilled water.
2. Materials and methods
2.2. Plant material and culture Pisum sativum cv. Sparkle (Rogers Bros. Seed Co., Twin falls, ID) is an early maturing freezer pea related to the parental line, Lincoln, (Boyce Thompson Institute, Ithaca, NY). The parental line, Lincoln, was used as a control, because A. rhizogenes did not induce roots on the parental line, Sparkle. Mutant lines were R25 (sym 8) and R72 (sym 9), non-allelic non-nodulating (Nod-) derivatives of Sparkle, obtained by 3' irradiation [7]. Both mutants produce root exudates that induce the nod genes of Rhizobium, and their roots are colonized normally by the bacterium [7]. However, they are defective in nodulation, i.e. root hair curling, infection and nodule meristem formation [7]. Thus, these mutants appear to be unresponsive to the nod factors produced by Rhizobium. Another mutant line tested was E 135 (sym 13), obtained by ethylmethane sulfonic acid treatment of Sparkle. This mutant is infected by Rhizobium, but forms white, early senesing nodules that lack leghemoglobin and nitrogenase activity [8], and is designated Nod +, Fix-. Plants were grown in a growth chamber with 16 h/8 h alternating day/night periods, a light intensity of 45/~E/m2/s and day and night temperatures of 27° and 18°C, respectively.
2.1. Bacterial culture Six strains of A. rhizogenes were tested for root induction on pea: A4 (L. Moore, Oregon State University), ATCC 15834 and ATCC 11325 (CRBF, Universit~ Laval), 8196, 2659 and 1855 (INRA, Versailles). Bacteria were maintained at 25°C on solid Yeast Mannitol Agar medium containing, in 1 1 of distilled water: 0.5 g K2HPO4, 2 g MgSO4.7H20, 0.1 g NaCI, 10 g mannitol, and 0.4 g yeast extract. The pH was adjusted to 7.0 before sterilization at 121°C for 16 min. Bacto
2.3. Seed germination, inoculation and root culture Seeds were surface-sterilized by immersion in 70% ethanol for 10 min, followed by 7% calcium hypochlorite for 10-15 min. They were then rinsed three times with sterile distilled water, and placed aseptically on 1% water agar for germination. Pregerminated seeds were transferred to Petri dishes [18] containing modified White's medium supplemented with sucrose (30.0 g/l). The medium contained in 1 1 of distilled water: 731 mg MgSO4.7H20, 453 mg Na2SO4, 80 mg KNO 3, 65
B. Balaji et al./ Plant Sci. 102 (1994) 195-203
mg KCI, 19 mg NaH2PO4.2H20, 288 mg Ca(NO3)2.4H20, 8 mg NaFe EDTA (ethylenediaminetetraacetic acid), 0.75 mg Kl, 6 mg MnCI2.4H20 , 2.65 mg ZnSO4.7H20, 1.5 mg H3BO3, 0.13 mg CuSO4.5H20, 0.0024 mg Na2MoO4.2H20, 3 mg glycine, 0.1 mg thiamine, 50 mg myo-inositol, and 30 g/l sucrose. The pH was adjusted to 5.5 before sterilization at 121°C for 16 min. The medium was solidified with 0.3% Gellan Gum (Gel-gro, ICN Biochemicals, Cleveland, OH). After 7-10 days, epicotyls were either wounded and inoculated with a sterile syringe needle containing the A. rhizogenes liquid inoculum or cut into segments and immersed for 45 s in the A. rhizogenes liquid inoculum. The epicotyl sections were then cultured on White's medium in Magenta vessels and incubated in the dark at 23°C. Roots appeared after 2-3 weeks. They were excised and placed on White's medium supplemented with the antibiotics, cefotaxime (0.2 g/l) (Sigma Chemical Co., St. Louis, MO) and carbenicillin (0.5 g/l) (Sigma) until visible contamination by Agrobacterium was eliminated, then roots were routinely subcuitured without antibiotics. Root apices (2-3 cm) from the seedlings of all the mutant lines and a parental line, Lincoln, were excised and cultured in White's medium to test the growth of normal (non-transformed) roots.
2.4. Fungal inoculum Spores of Gigaspora margarita Becker & Hall (DAOM 194757, deposited at the Biosystematic Research Center, Ottawa)-were isolated from a leek (Allium Porrum L) pot culture by wet sieving [19] followed by a density gradient centrifugation [20]. Spores were surface-sterilized with 2% chloramine T, rinsed in an antibiotic solution (containing 1% gentamycin sulfate and 2% streptomycin sulfate) [4] and stored on water agar at 4°C until used. Healthy white to cream-colored spores were selected with the aid of a stereo microscope and aseptically transferred from agar plates with a scalpel blade. For inoculation, one spore per Petri plate was used as an experimental unit. 2.5. DNA isolation and molecular hybridization High molecular weight genomic DNA was isola-
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ted from 2 g of 10-day-old roots following the methods of Davis et al. [21] and Dellaporta et al. [22] with some modifications. Roots were ground to fine powder in liquid nitrogen and incubated in 10 ml of lysis buffer, containing 50 mM Tris-HC1 (pH 7.5), 100 mM NaCI, 50 mM EDTA (pH 8.0), 0.5% sodium dodecylsulfate and 20 mM /3mercaptoethanol, for 10 min at room temperature and centrifuged for 8 min at 13 000 rev./min. Extraction with two volumes of phenol:chloroform:isoamyl alcohol (25:24:1)was followed by centrifugation at 13 000 rev./min for 8 min. Genomic DNA was precipitated by the addition of 1 volume of cold isopropyl alcohol to the supernatant fraction. The precipitated DNA was then dissolved in TE buffer, containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, and stored at -20°C until used. Genomic DNA samples (10 #g) were digested with 50 units of EcoRl at 37°C overnight, subjected to electrophoresis and transferred to a Hybond-N hybridization transfer membrane [23]. Transfer and hybridization were as recommended by the manufacturer (Amersham). The probe was pLJ1, which represents the entire Ri TL-DNA [24]. Labeling was by random priming (Amersham). The filter was washed with 2 x SSC, 0.1% (w/v) SDS at room temperature for 10 min and subsequently with 1 x SSC, 0.1% (w/v) SDS for 15 min and 0.1 xSSC, 0.1% (w/v) SDS for 15 min at 65°C. Autoradiography was with Kodak XOMAT scientific imaging film for 10 days.
2.6. Culture of transformed roots Transformed roots were routinely maintained on modified White's medium solidified with 0.3% Gellan Gum, subculturing every 15 days. This medium did not favor the growth of VAM spores in dual culture on transformed roots, so minimal medium was used [4], containing, in 1 1 of distilled water: 731 mg MgSO4.7H20, 80 mg KNO3, 65 mg KCI, 4.8 mg KH2PO4, 288 mg Ca(NO3)2.4H20, 10 g sucrose, 8 mg NaFe EDTA, 0.75 mg KI, 6 mg MnC12.H20, 2.65 mg ZnSOa.7H20, 1.5 mg H3BO3, 0.13 mg CuSO4.5H20, 0.0024 mg NaEMoO4.2H20, 3 mg glycine, 0.1 mg thiamine hydrochloride, 0.1 mg pyridoxine hydrochloride, 0.5 mg nicotinic acids and 50 mg myo-inositol. The
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pH was adjusted to 5.5 before sterilization at 121°C for 16 min. The medium was solidified with 0.4% Gellan gum (Gel-gro, I C N Biochemicals, Cleveland, OH). Roots were used to study interactions with G. margarita 22 days after subculture. 2. 7. Establishment o f dual ( V A M / r o o t ) culture A single pre-germinated spore placed in a Petri dish with a single transformed root served to initiate the interaction. Dishes were sealed with Parafilm and incubated in a vertical position with the spores on the bottom. The extent of VAM interaction with the host was observed periodically using a dissecting microscope and light and electron microscopy. Samples were prepared for light microscopy by clearing them in 10% K O H (w/v) for 5 min in an autoclave at 121°C. They were rinsed three times with sterile distilled water to remove excess K O H and stained in 0.1 (w/v) chlorazol black E for 3 min at 121°C in the autoclave. Individual root segments were mounted on a glass slide and observed for mycorrhizal colonization. Other samples were fixed overnight in 2.0% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4°C and rinsed in the same buffer. Samples were postfixed for 1 h in 2% osmium tetroxide and rinsed in distilled water. They were dehydrated through an acetone series followed by three changes of pure acetone, infiltrated in an acetone-Spurr's resin series and embedded in 100% Spurr's resin. Polymerization took place at 70°C for 48 h. For light microscopy, transverse sections, approximately 0.5-1.0 #m thick, were cut from the embedded tissue and stained with 0.05% toluidine blue 0 in 1.0% sodium tetraborate (Borax). For transmission electron microscopy (TEM), 60-80 nm sections were mounted on copper grids and treated with uranyl acetate for 15 min [25] and with lead citrate for another 15 min to improve contrast [26]. For each treatment, at least 20 root specimens were examined using a Leitz Ortholux II light microscope, and another 20 specimens were viewed in a Siemens Elmiskop 103 electron microscope. Samples were taken 14 and 21 days after the first contact between the roots and the growing hyphae.
3. Results 3.1. Callus formation All strains of A. rhizogenes induced callus formation 7 days after inoculation of pea epicotyls (Fig. 1). Adventitious roots appeared 10 days after inoculation on callus induced by A. rhizogenes strains ATCC 15834, 2659 and 1855 (Fig. 2). All transformed roots of mutants and the parental line, Lincoln, induced by A. rhizogenes strain 1855, grew fast and were highly branched (Figs. 3 and 4). In contrast, normal (non-transformed) roots, excised from seedlings of all the mutants and the parental line, Lincoln, (Fig. 5), elongated slowly and
Fig. I. Epicotyl segment of parental line, Lincoln, showing the formation of callus (C) at the inoculated site after 7 days. Abbreviation: C, callus. Bar = 2 mm. Fig. 2. Formation of callus and adventitious roots on a decapitated epicotyl of parental line, Lincoln, 14 days after inoculation. Abbreviation: C, callus. Bar = 2 mm. Fig. 3. Adventitious root growth (e.g., arrow) from inoculated segments of the parental line, Lincoln, 21 days after inoculation. Bar = 3.3 cm. Fig. 4. Transformed roots of the parental line, Lincoln, 15 days after culture in modified White's medium. Bar = 3.3 cm. Fig. 5. Normal roots (non-transformed) of the parental line, Lincoln, 15 days after culture in modified White's medium. Bar = 3.3 cm.
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I
I
I
I
I
I
EcoR1 15
EcoR1 36 EcoR1 37a,b EcoR1 40
Ri TL-DNA (EcoR1) 1.0 kb internal fragments Fig. 6. Southern hybridization after digestion of pea DNA with fragments are differentiated by cross-hatching.
Table 1 Symbiotic characteristics of Host
cv. Sparkle mutants R25 R72 Ei35 Parental Lincoln
Pisum sativum
~ EcoRl
border fragments and hybridization with the probe pLJI. Border and internal
L. cv. Sparkle mutants and parental line, Lincoln
Gene conditioning symbiosis
Symbiotic characters Nodulation (Nod)
Fixation a (Fix)
Mycorrhization (Myc)
sym 8
-
-
-
sym 9
-
-
-
+ +
+
+ +
sym
13
normal symbiotic interaction; - , lack of symbiotic interaction. aNitrogen fixation phenotype, according to Refs. [7,8].
Symbols." +,
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B. Balaji et al. / Plant Sci. 102 (1994) 195-203 stopped growing after 7 days, when cultured in W h i t e ' s medium. Surprisingly, the p a r e n t a l line, Sparkle, did not react f a v o r a b l y to inoculation by A. rhizogenes. Calli were p r o d u c e d that failed to form roots. Therefore, roots from the related line, Lincoln, were used as controls. The c o m b i n a t i o n o f cefotaxime (0.2 g/l) a n d carbenicillin (0.5 g/l) was effective in eliminating bacterial c o n t a m i n a tion o f roots. 3.2. Confirmation o f transformation D N A from all the t r a n s f o r m e d roots o f the m u t a n t s and the p a r e n t a l line, Lincoln, hybridized well to the p r o b e representing the T L - D N A (pLJ 1) (Fig. 6), and no h y b r i d i z a t i o n signal was observed in the D N A from the n o r m a l roots. Putative b o r d e r fragments, representing j u n c t i o n s between the foreign a n d host D N A , varied from line to line. Five internal fragments from the T L - D N A ( E c o R l 15, EcoR1 36, E c o R l 37a, b a n d EcoR1 40) were recognized by their intensity a n d m o l e c u l a r weight. G e n o m i c D N A from t r a n s f o r m e d a n d normal roots was subjected to P C R , using primers representing the 5 ' a n d 3 ' ends o f the rolA c o d i n g sequence. T r a n s f o r m e d roots, but n o t the n o r m a l roots, showed a b a n d c o r r e s p o n d i n g to the rolA gene, confirming t r a n s f o r m a t i o n (results not shown).
Fig. 7. Longitudinal section of El3 ~; (sym 13) after squashing and staining showing colonization by G. margarita. Arbuscules (single arrowhead) are conspicuous. Bar = 1.0 mm. Fig. 8. Longitudinal section of R25 (sym 8) after squashing and staining, showing the mycelium (arrowhead) growing on the root surface. Bar = 1.0 m m . Fig. 9. Light micrograph of a transverse section of R72 (sym 9) 14 days after contact with G. margarita, showing lack of invasion. Abbreviation: Co, cortex. Bar = 0.5 mm. Fig. 10. Light micrograph of a transverse section of E 135 (sym 13) 14 days after exposure to G. margarita showing arbuscules (Ar) in the colonized cortex, surrounded by a few non-infected zones. Abbreviation: Ar, arbuscules. Bar = 0.5 mm.
3.3. Dual transformed r o o t / V A M culture M y c o r r h i z a t i o n , n o d u l a t i o n a n d nitrogen fixation phenotypes [7,81 are s u m m a r i z e d in T a b l e 1. All the n o d u l a t i n g p h e n o t y p e s (Nod+), whether Fix + or F i x - , f o r m e d V A m y c o r r h i z a s (Myc+), whereas the m u t a n t s unable to form nodules ( N o d - ) were also unable to form m y c o r r h i z a s (Myc-).
Fig. 11. Transmission electron micrograph (TEM) of a transverse section of R72 (sym 9), showing the epidermis resisting the entry of G. margarita hypha (H) (single arrowheads) after 21 days of contact between the fungus and the growing roots. Abbreviations: E, epidermis; H, hypha. Bar = 200 nm. Fig. 12. TEM of a transverse section of a cortical cell of E135 (sym 13) showing a penetrating hyphal structure (Hp) attempting to colonize the adjacent cell, 21 days after contact between the fungus and the growing roots. Abbreviations: Ar, arbuscules; H, hypha. Bar = 2 p.m.
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Light and electron microscopy were used to determine the nature of the r o o t - V A M interaction. Squashed and stained longitudinal sections of E135 (sym 13) (Nod +, Fix-) revealed extensive colonization by G. margarita (Fig. 7), with evident arbuscular structures. Transformed roots of the parental line, Lincoln, showed a similar colonization pattern to that of E135 (sym 13). Longitudinal sections (Fig. 8) of R25 (sym 8) (Nod-, Fix-) and R72 (sym 9) (Nod-, Fix-) (not shown) showed mycelium outside the roots, but not within the tissues. Using light microscopic examination of transverse sections of R25 (sym 8) and R72 (sym 9) (Nod-, Fix-) roots fixed after 14 days of contact with the hypha of G. margarita, we failed to detect either penetration of the epidermis or colonization of the cortex (Fig. 9). In contrast, similar examination of E135 (sym 13) (Nod +, Fix-) (Fig. 10) clearly showed the epidermal entry point and invasion of the cortex, with arbuscule formation. Noninfected cortical cells were seen between the colonized cells. Transmission electron microscopy (TEM) of a transverse section of R72 (sym 9) (Fig. 11) after 21 days of contact between the fungus and the growing roots confirmed that invasion had not taken place, but that G. margarita had tried to enter the epidermis (Fig. 11). TEM of transverse sections of E135 (sym 13) (Fig. 12) showed after 21 days of contact between the roots and hyphae that a cortical cell had been completely filled with arbuscules. A hypha had also formed a tube-like structure to enter the adjacent cell. The parental line, Lincoln, was similarly colonized. 4. Discussion
The phenotypes of transformed roots were similar to those already reported for pea [27]. They were better adapted to growth in culture than normal roots, growing faster and surviving for longer periods without subculture [151. The difficulty in transforming Sparkle was overcome in later (unpublished) work, and is probably not due to the genetic makeup of this variety. Fortunately, we were able to use a related line, Lincoln, as a control. Molecular hybridization showed that EcoR1
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fragments corresponding to the core TL-DNA [28] were inserted into all 4 root clones. Border fragments varied from clone to clone, as expected for independent insertions. The two Nod- mutants examined, R25 (sym 8) and R72 (sym 9) were also Myc- in vitro (Table 1). We did not find the anomalies reported previously [5]: a mutant faba bean, cv. Ascott F48, was Nod- and Myc+; cv. Indian 778 was Nod + and Myc-, and other P. sativum, cv. Frisson, mutants (F4.122, F4.-58, F4-218 P5, P7, P9 and PI0) were also Nod-, Myc +. Duc et al. [5] also observed occasional arbuscules in a Nod- pea mutant. They attributed this deviation to a breakdown of resistance to mycorrhizal colonization. In our study, Myc and Nod phenotypes were similar. In the Nod-, Fix- mutants we have considered, G. margarita failed to invade the root cortex, but colonized the root surface, making contact with the epidermis. We noted one case in which a hypha formed a beak-like structure (Fig. 11) in an apparent attempt to penetrate the root, raising the question of the nature of the barriers to fungal invasion of the root interior in the Nod- mutants. We suppose that structural barriers or deficient signals halted the process of infection. Markwei and LaRue [7] observed no structural modification of the epidermis when the Myc- mutant roots were treated with Rhizobium, but noted epidermal thickening after they were exposed to pathogenic Pythium spp. We also did not see modification of the epidermis in either the mutants or the controls after contact with G. margarita. Thus, if epidermal thickening is a defense reaction, it does not explain the resistance of the mutants to VAM invasion. Since these mutants do not permit penetration by Rhizobium, showing no root hair curling, it would seem likely that they fail to perceive and react to signals necessary to the interaction. Until now, Myc- genotypes have been detected in symbiotic mutants of three commercially important legume species, faba beans, peas [5] and alfalfa [6]. The correlation between the Nod- and Myc- phenotypes raises the possibility that VAM fungi rely on the same signals as Rhizobium to initiate a favorable reaction from the plant. There is no fossil record to indicate when nodulation and mycorrhization evolved, but we
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can speculate that the use of a chitin-like glucosamine nod factors by Rhizobium and VAM fungi was derived from an ancestral plant/ microorganism interaction [29]. This hypothesis can be pursued by attempting to find homologues to nod genes in VAM fungi. The practical problem of culturing VAM fungi remains, but if useful information can be gained from Rhizobium [30] concerning the basic nature of the interaction, culture of VAM fungi may be facilitated. Furthermore, transformed roots can be cultured on a large scale, making it possible to collect large quantities of root exudate, which should aid in the search for substances produced by roots that promote the growth of VAM fungi.
Acknowledgments We wish to thank Dr Horst Vierheilig, Dr Preman Singh and Paul de la Bastide, Universit6 Laval for their critical reviews of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council (Canada) through a grant to Y. Pich6 and by the Cooperation France-Quebec to D. Tepfer and Y. Pich6. B. Balaji acknowledges CIES (Centre International des Etudiants et Stagiaires) for financial assistance during his stay in France.
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B. Balaji et al./ Plant Sci. 102 (1994) 195-203 [24] L. Jouanin, Restriction map of an agropine-type Ri plasmid and its homologies with Ti plasmids. Plasmid, 12 (1984) 91-102. [25] R.C. Valentines, Contrast enhancement in the electron microscopy of viruses. Adv. Virus Res., 8 (1961) 287-318. [26] E.S. Reynolds, The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17 (1963) 208-212. [27] J. Bercetche, D. Chriqui, S. Adam and C. David, Morphogenetic and cellular reorientations induced by Agrobacterium rhizogenes (Strains 1855, 2659 and 8196) on carrot, pea and tobacco. Plant Sci., 52 (1987) 195-210.
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[28] L.-Y. Sun, G. Touraud, C. Charbonnier and D. Tepfer, Modification of phenotype in Belgian endive (Cichorium intybus) through genetic transformation by Agrobacterium rhizogenes: conversion from biennial to annual flowering. Transgenic Res., 1 (1991) 14-22. [29] L. Simon, J. Bousquet, R.C. L6vesque and M. Lalonde, Origin and diversification of endomycorrhizal fungi and coincidence with vascular land plants. Nature, 363 (1993) 67-69. [30] S.R. Long, Rhizobium-legume nodulation: life together in the underground. Cell, 56 (1989) 203-214.
Plant Science 102 (1994) 195-203
Pisum sativum mutants insensitive to nodulation are also insensitive to invasion in vitro by the mycorrhizal fungus,
Gigaspora margarita Boovaraghan
B a l a j i *a, A m a d o u
M . B a a'l, T h o m a s Yves Pich6 a
A. L a R u e b, D a v i d T e p f e r c,
aCentre de Recherche en Biologie Forestibre, Facult~ de Foresterie et de G~omatique, Universit~ Laval, Quebec, Quebec, Canada GIK 7P4 bBoyce Thompson Institute for Plant Research, Tower Road, Ithaca, N Y 14853-1801, USA CLaboratoire de Biologie de la Rhizosph~re, lnstitut National de la Recherche Agronomique, Versailles Cedex, F-78026, France Received 8 July 1994; accepted 3 August 1994
Abstract
Transformed root cultures were established using Agrobacterium rhizogenes inoculation of pea mutants, altered in their interaction with Rhizobium. They were tested in an in vitro model for sensitivity to the vesicular-arbuscular mycorrhizal (VAM) fungus, Gigasporamargarita. VAM development was assessed using light and electron microscopy. Two non-nodulating, non-nitrogen fixing (Nod-, Fix-) pea mutants were resistant to VAM colonization in vitro: Mycelium developed on the root surface but failed to colonize the interior. A nodulating (Nod +) genotype, which was unable to fix nitrogen (Fix-) in association with Rhizobium and a parental line, Lincoln (Nod +, Fix+), interacted normally with the fungus, showing extensive internal colonization. These results confirm, under axenic conditions, previous reports showing that defective nodulation is correlated with defective mycorrhization. We propose using this in vitro model to identify factors necessary to initiating and maintaining the VAM/plant symbiosis.
Keywords: VAM; Myc mutants; Pisum sativum; Gigaspora margarita; Agrobacterium rhizogenes; Rhizobium; Genetic transformation
1. Introduction
Vesicular-arbuscular mycorrhizas (VAM) are ubiquitous and associate with most of the vascular * Corresponding author. I Present Address: Institut de Recherche en Biologie et Ecologie Tropicale, BP 7047 Ouagadougou 03, Burkina Faso, West Africa.
plants [1-3], for which they provide essential nutrients and protect againr * drought and plant pathogens. Among 202 species of legumes surveyed, almost 90% engaged in mycorrhizal symbioses, and among these more than 82% were of the VAM type [1]; however, cultures of VAM fungi have not been obtained in the absence of plant host [4], impeding comprehension and manipulation of the symbiosis. The role of the host
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(94)03964-B
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plant, although easier to study, has only recently received attention. Even though there has not been an exhaustive genetic study of competence by legumes to establishing VAM symbiosis, mutants resistant to mycorrhization by the fungus were reported [5,6], and this defect was correlated with defective nodulation by Rhizobium [7]. We performed similar studies using an in vitro model for the VAM/root interaction, in which mutants of Pisum sativum that had been designated either capable (Nod÷) or incapable (Nod-) of establishing VAM symbiosis [7,8] were infected with the VAM fungus, Gigaspora margarita, in the absence of contaminating microorganisms. We thus verified that the Myc- phenotype is correlated with the inability to nodulate (Nod-), but is not correlated with the inability to fix nitrogen (Fix-) in association with Rhizobium. The experimental system made use of transformed root organ cultures, produced by inoculating different pea genotypes with A. rhizogenes. Although normal VAM interactions can take place in axenically grown plants in agar [9-11] or on non-transformed root organ cultures [12], transformed roots grow faster than normal roots [13] and provide a reproducible and simple system for studying VAM symbioses [14-171. This paper reports the isolation of transformed roots from Myc + and Myc- mutants of pea (P. sativum cv. Sparkle) and their interaction with a VAM fungus, Gigaspora margarita, in vitro. Three transformed lines and a parental line, Lincoln, were examined for their response to the mycorrhizal fungus G. margarita.
Agar was included at 15.0 g/l, (Difco Laboratories, Detroit, MI). The same medium without agar (5 ml) was used to culture bacteria for 48 h prior to plant inoculation using a rotary shaker at 100 rev./min at 27°C. Inoculum was prepared by centrifuging bacteria for 10 min at 1775 x g and resuspending them in 20 ml of sterile distilled water.
2. Materials and methods
2.2. Plant material and culture Pisum sativum cv. Sparkle (Rogers Bros. Seed Co., Twin falls, ID) is an early maturing freezer pea related to the parental line, Lincoln, (Boyce Thompson Institute, Ithaca, NY). The parental line, Lincoln, was used as a control, because A. rhizogenes did not induce roots on the parental line, Sparkle. Mutant lines were R25 (sym 8) and R72 (sym 9), non-allelic non-nodulating (Nod-) derivatives of Sparkle, obtained by 3' irradiation [7]. Both mutants produce root exudates that induce the nod genes of Rhizobium, and their roots are colonized normally by the bacterium [7]. However, they are defective in nodulation, i.e. root hair curling, infection and nodule meristem formation [7]. Thus, these mutants appear to be unresponsive to the nod factors produced by Rhizobium. Another mutant line tested was E 135 (sym 13), obtained by ethylmethane sulfonic acid treatment of Sparkle. This mutant is infected by Rhizobium, but forms white, early senesing nodules that lack leghemoglobin and nitrogenase activity [8], and is designated Nod +, Fix-. Plants were grown in a growth chamber with 16 h/8 h alternating day/night periods, a light intensity of 45/~E/m2/s and day and night temperatures of 27° and 18°C, respectively.
2.1. Bacterial culture Six strains of A. rhizogenes were tested for root induction on pea: A4 (L. Moore, Oregon State University), ATCC 15834 and ATCC 11325 (CRBF, Universit~ Laval), 8196, 2659 and 1855 (INRA, Versailles). Bacteria were maintained at 25°C on solid Yeast Mannitol Agar medium containing, in 1 1 of distilled water: 0.5 g K2HPO4, 2 g MgSO4.7H20, 0.1 g NaCI, 10 g mannitol, and 0.4 g yeast extract. The pH was adjusted to 7.0 before sterilization at 121°C for 16 min. Bacto
2.3. Seed germination, inoculation and root culture Seeds were surface-sterilized by immersion in 70% ethanol for 10 min, followed by 7% calcium hypochlorite for 10-15 min. They were then rinsed three times with sterile distilled water, and placed aseptically on 1% water agar for germination. Pregerminated seeds were transferred to Petri dishes [18] containing modified White's medium supplemented with sucrose (30.0 g/l). The medium contained in 1 1 of distilled water: 731 mg MgSO4.7H20, 453 mg Na2SO4, 80 mg KNO 3, 65
B. Balaji et al./ Plant Sci. 102 (1994) 195-203
mg KCI, 19 mg NaH2PO4.2H20, 288 mg Ca(NO3)2.4H20, 8 mg NaFe EDTA (ethylenediaminetetraacetic acid), 0.75 mg Kl, 6 mg MnCI2.4H20 , 2.65 mg ZnSO4.7H20, 1.5 mg H3BO3, 0.13 mg CuSO4.5H20, 0.0024 mg Na2MoO4.2H20, 3 mg glycine, 0.1 mg thiamine, 50 mg myo-inositol, and 30 g/l sucrose. The pH was adjusted to 5.5 before sterilization at 121°C for 16 min. The medium was solidified with 0.3% Gellan Gum (Gel-gro, ICN Biochemicals, Cleveland, OH). After 7-10 days, epicotyls were either wounded and inoculated with a sterile syringe needle containing the A. rhizogenes liquid inoculum or cut into segments and immersed for 45 s in the A. rhizogenes liquid inoculum. The epicotyl sections were then cultured on White's medium in Magenta vessels and incubated in the dark at 23°C. Roots appeared after 2-3 weeks. They were excised and placed on White's medium supplemented with the antibiotics, cefotaxime (0.2 g/l) (Sigma Chemical Co., St. Louis, MO) and carbenicillin (0.5 g/l) (Sigma) until visible contamination by Agrobacterium was eliminated, then roots were routinely subcuitured without antibiotics. Root apices (2-3 cm) from the seedlings of all the mutant lines and a parental line, Lincoln, were excised and cultured in White's medium to test the growth of normal (non-transformed) roots.
2.4. Fungal inoculum Spores of Gigaspora margarita Becker & Hall (DAOM 194757, deposited at the Biosystematic Research Center, Ottawa)-were isolated from a leek (Allium Porrum L) pot culture by wet sieving [19] followed by a density gradient centrifugation [20]. Spores were surface-sterilized with 2% chloramine T, rinsed in an antibiotic solution (containing 1% gentamycin sulfate and 2% streptomycin sulfate) [4] and stored on water agar at 4°C until used. Healthy white to cream-colored spores were selected with the aid of a stereo microscope and aseptically transferred from agar plates with a scalpel blade. For inoculation, one spore per Petri plate was used as an experimental unit. 2.5. DNA isolation and molecular hybridization High molecular weight genomic DNA was isola-
197
ted from 2 g of 10-day-old roots following the methods of Davis et al. [21] and Dellaporta et al. [22] with some modifications. Roots were ground to fine powder in liquid nitrogen and incubated in 10 ml of lysis buffer, containing 50 mM Tris-HC1 (pH 7.5), 100 mM NaCI, 50 mM EDTA (pH 8.0), 0.5% sodium dodecylsulfate and 20 mM /3mercaptoethanol, for 10 min at room temperature and centrifuged for 8 min at 13 000 rev./min. Extraction with two volumes of phenol:chloroform:isoamyl alcohol (25:24:1)was followed by centrifugation at 13 000 rev./min for 8 min. Genomic DNA was precipitated by the addition of 1 volume of cold isopropyl alcohol to the supernatant fraction. The precipitated DNA was then dissolved in TE buffer, containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, and stored at -20°C until used. Genomic DNA samples (10 #g) were digested with 50 units of EcoRl at 37°C overnight, subjected to electrophoresis and transferred to a Hybond-N hybridization transfer membrane [23]. Transfer and hybridization were as recommended by the manufacturer (Amersham). The probe was pLJ1, which represents the entire Ri TL-DNA [24]. Labeling was by random priming (Amersham). The filter was washed with 2 x SSC, 0.1% (w/v) SDS at room temperature for 10 min and subsequently with 1 x SSC, 0.1% (w/v) SDS for 15 min and 0.1 xSSC, 0.1% (w/v) SDS for 15 min at 65°C. Autoradiography was with Kodak XOMAT scientific imaging film for 10 days.
2.6. Culture of transformed roots Transformed roots were routinely maintained on modified White's medium solidified with 0.3% Gellan Gum, subculturing every 15 days. This medium did not favor the growth of VAM spores in dual culture on transformed roots, so minimal medium was used [4], containing, in 1 1 of distilled water: 731 mg MgSO4.7H20, 80 mg KNO3, 65 mg KCI, 4.8 mg KH2PO4, 288 mg Ca(NO3)2.4H20, 10 g sucrose, 8 mg NaFe EDTA, 0.75 mg KI, 6 mg MnC12.H20, 2.65 mg ZnSOa.7H20, 1.5 mg H3BO3, 0.13 mg CuSO4.5H20, 0.0024 mg NaEMoO4.2H20, 3 mg glycine, 0.1 mg thiamine hydrochloride, 0.1 mg pyridoxine hydrochloride, 0.5 mg nicotinic acids and 50 mg myo-inositol. The
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pH was adjusted to 5.5 before sterilization at 121°C for 16 min. The medium was solidified with 0.4% Gellan gum (Gel-gro, I C N Biochemicals, Cleveland, OH). Roots were used to study interactions with G. margarita 22 days after subculture. 2. 7. Establishment o f dual ( V A M / r o o t ) culture A single pre-germinated spore placed in a Petri dish with a single transformed root served to initiate the interaction. Dishes were sealed with Parafilm and incubated in a vertical position with the spores on the bottom. The extent of VAM interaction with the host was observed periodically using a dissecting microscope and light and electron microscopy. Samples were prepared for light microscopy by clearing them in 10% K O H (w/v) for 5 min in an autoclave at 121°C. They were rinsed three times with sterile distilled water to remove excess K O H and stained in 0.1 (w/v) chlorazol black E for 3 min at 121°C in the autoclave. Individual root segments were mounted on a glass slide and observed for mycorrhizal colonization. Other samples were fixed overnight in 2.0% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4°C and rinsed in the same buffer. Samples were postfixed for 1 h in 2% osmium tetroxide and rinsed in distilled water. They were dehydrated through an acetone series followed by three changes of pure acetone, infiltrated in an acetone-Spurr's resin series and embedded in 100% Spurr's resin. Polymerization took place at 70°C for 48 h. For light microscopy, transverse sections, approximately 0.5-1.0 #m thick, were cut from the embedded tissue and stained with 0.05% toluidine blue 0 in 1.0% sodium tetraborate (Borax). For transmission electron microscopy (TEM), 60-80 nm sections were mounted on copper grids and treated with uranyl acetate for 15 min [25] and with lead citrate for another 15 min to improve contrast [26]. For each treatment, at least 20 root specimens were examined using a Leitz Ortholux II light microscope, and another 20 specimens were viewed in a Siemens Elmiskop 103 electron microscope. Samples were taken 14 and 21 days after the first contact between the roots and the growing hyphae.
3. Results 3.1. Callus formation All strains of A. rhizogenes induced callus formation 7 days after inoculation of pea epicotyls (Fig. 1). Adventitious roots appeared 10 days after inoculation on callus induced by A. rhizogenes strains ATCC 15834, 2659 and 1855 (Fig. 2). All transformed roots of mutants and the parental line, Lincoln, induced by A. rhizogenes strain 1855, grew fast and were highly branched (Figs. 3 and 4). In contrast, normal (non-transformed) roots, excised from seedlings of all the mutants and the parental line, Lincoln, (Fig. 5), elongated slowly and
Fig. I. Epicotyl segment of parental line, Lincoln, showing the formation of callus (C) at the inoculated site after 7 days. Abbreviation: C, callus. Bar = 2 mm. Fig. 2. Formation of callus and adventitious roots on a decapitated epicotyl of parental line, Lincoln, 14 days after inoculation. Abbreviation: C, callus. Bar = 2 mm. Fig. 3. Adventitious root growth (e.g., arrow) from inoculated segments of the parental line, Lincoln, 21 days after inoculation. Bar = 3.3 cm. Fig. 4. Transformed roots of the parental line, Lincoln, 15 days after culture in modified White's medium. Bar = 3.3 cm. Fig. 5. Normal roots (non-transformed) of the parental line, Lincoln, 15 days after culture in modified White's medium. Bar = 3.3 cm.
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I
I
I
I
I
I
EcoR1 15
EcoR1 36 EcoR1 37a,b EcoR1 40
Ri TL-DNA (EcoR1) 1.0 kb internal fragments Fig. 6. Southern hybridization after digestion of pea DNA with fragments are differentiated by cross-hatching.
Table 1 Symbiotic characteristics of Host
cv. Sparkle mutants R25 R72 Ei35 Parental Lincoln
Pisum sativum
~ EcoRl
border fragments and hybridization with the probe pLJI. Border and internal
L. cv. Sparkle mutants and parental line, Lincoln
Gene conditioning symbiosis
Symbiotic characters Nodulation (Nod)
Fixation a (Fix)
Mycorrhization (Myc)
sym 8
-
-
-
sym 9
-
-
-
+ +
+
+ +
sym
13
normal symbiotic interaction; - , lack of symbiotic interaction. aNitrogen fixation phenotype, according to Refs. [7,8].
Symbols." +,
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B. Balaji et al. / Plant Sci. 102 (1994) 195-203 stopped growing after 7 days, when cultured in W h i t e ' s medium. Surprisingly, the p a r e n t a l line, Sparkle, did not react f a v o r a b l y to inoculation by A. rhizogenes. Calli were p r o d u c e d that failed to form roots. Therefore, roots from the related line, Lincoln, were used as controls. The c o m b i n a t i o n o f cefotaxime (0.2 g/l) a n d carbenicillin (0.5 g/l) was effective in eliminating bacterial c o n t a m i n a tion o f roots. 3.2. Confirmation o f transformation D N A from all the t r a n s f o r m e d roots o f the m u t a n t s and the p a r e n t a l line, Lincoln, hybridized well to the p r o b e representing the T L - D N A (pLJ 1) (Fig. 6), and no h y b r i d i z a t i o n signal was observed in the D N A from the n o r m a l roots. Putative b o r d e r fragments, representing j u n c t i o n s between the foreign a n d host D N A , varied from line to line. Five internal fragments from the T L - D N A ( E c o R l 15, EcoR1 36, E c o R l 37a, b a n d EcoR1 40) were recognized by their intensity a n d m o l e c u l a r weight. G e n o m i c D N A from t r a n s f o r m e d a n d normal roots was subjected to P C R , using primers representing the 5 ' a n d 3 ' ends o f the rolA c o d i n g sequence. T r a n s f o r m e d roots, but n o t the n o r m a l roots, showed a b a n d c o r r e s p o n d i n g to the rolA gene, confirming t r a n s f o r m a t i o n (results not shown).
Fig. 7. Longitudinal section of El3 ~; (sym 13) after squashing and staining showing colonization by G. margarita. Arbuscules (single arrowhead) are conspicuous. Bar = 1.0 mm. Fig. 8. Longitudinal section of R25 (sym 8) after squashing and staining, showing the mycelium (arrowhead) growing on the root surface. Bar = 1.0 m m . Fig. 9. Light micrograph of a transverse section of R72 (sym 9) 14 days after contact with G. margarita, showing lack of invasion. Abbreviation: Co, cortex. Bar = 0.5 mm. Fig. 10. Light micrograph of a transverse section of E 135 (sym 13) 14 days after exposure to G. margarita showing arbuscules (Ar) in the colonized cortex, surrounded by a few non-infected zones. Abbreviation: Ar, arbuscules. Bar = 0.5 mm.
3.3. Dual transformed r o o t / V A M culture M y c o r r h i z a t i o n , n o d u l a t i o n a n d nitrogen fixation phenotypes [7,81 are s u m m a r i z e d in T a b l e 1. All the n o d u l a t i n g p h e n o t y p e s (Nod+), whether Fix + or F i x - , f o r m e d V A m y c o r r h i z a s (Myc+), whereas the m u t a n t s unable to form nodules ( N o d - ) were also unable to form m y c o r r h i z a s (Myc-).
Fig. 11. Transmission electron micrograph (TEM) of a transverse section of R72 (sym 9), showing the epidermis resisting the entry of G. margarita hypha (H) (single arrowheads) after 21 days of contact between the fungus and the growing roots. Abbreviations: E, epidermis; H, hypha. Bar = 200 nm. Fig. 12. TEM of a transverse section of a cortical cell of E135 (sym 13) showing a penetrating hyphal structure (Hp) attempting to colonize the adjacent cell, 21 days after contact between the fungus and the growing roots. Abbreviations: Ar, arbuscules; H, hypha. Bar = 2 p.m.
B. Balaji et al./Plant Sci.; 102 (!994) 195-203
Light and electron microscopy were used to determine the nature of the r o o t - V A M interaction. Squashed and stained longitudinal sections of E135 (sym 13) (Nod +, Fix-) revealed extensive colonization by G. margarita (Fig. 7), with evident arbuscular structures. Transformed roots of the parental line, Lincoln, showed a similar colonization pattern to that of E135 (sym 13). Longitudinal sections (Fig. 8) of R25 (sym 8) (Nod-, Fix-) and R72 (sym 9) (Nod-, Fix-) (not shown) showed mycelium outside the roots, but not within the tissues. Using light microscopic examination of transverse sections of R25 (sym 8) and R72 (sym 9) (Nod-, Fix-) roots fixed after 14 days of contact with the hypha of G. margarita, we failed to detect either penetration of the epidermis or colonization of the cortex (Fig. 9). In contrast, similar examination of E135 (sym 13) (Nod +, Fix-) (Fig. 10) clearly showed the epidermal entry point and invasion of the cortex, with arbuscule formation. Noninfected cortical cells were seen between the colonized cells. Transmission electron microscopy (TEM) of a transverse section of R72 (sym 9) (Fig. 11) after 21 days of contact between the fungus and the growing roots confirmed that invasion had not taken place, but that G. margarita had tried to enter the epidermis (Fig. 11). TEM of transverse sections of E135 (sym 13) (Fig. 12) showed after 21 days of contact between the roots and hyphae that a cortical cell had been completely filled with arbuscules. A hypha had also formed a tube-like structure to enter the adjacent cell. The parental line, Lincoln, was similarly colonized. 4. Discussion
The phenotypes of transformed roots were similar to those already reported for pea [27]. They were better adapted to growth in culture than normal roots, growing faster and surviving for longer periods without subculture [151. The difficulty in transforming Sparkle was overcome in later (unpublished) work, and is probably not due to the genetic makeup of this variety. Fortunately, we were able to use a related line, Lincoln, as a control. Molecular hybridization showed that EcoR1
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fragments corresponding to the core TL-DNA [28] were inserted into all 4 root clones. Border fragments varied from clone to clone, as expected for independent insertions. The two Nod- mutants examined, R25 (sym 8) and R72 (sym 9) were also Myc- in vitro (Table 1). We did not find the anomalies reported previously [5]: a mutant faba bean, cv. Ascott F48, was Nod- and Myc+; cv. Indian 778 was Nod + and Myc-, and other P. sativum, cv. Frisson, mutants (F4.122, F4.-58, F4-218 P5, P7, P9 and PI0) were also Nod-, Myc +. Duc et al. [5] also observed occasional arbuscules in a Nod- pea mutant. They attributed this deviation to a breakdown of resistance to mycorrhizal colonization. In our study, Myc and Nod phenotypes were similar. In the Nod-, Fix- mutants we have considered, G. margarita failed to invade the root cortex, but colonized the root surface, making contact with the epidermis. We noted one case in which a hypha formed a beak-like structure (Fig. 11) in an apparent attempt to penetrate the root, raising the question of the nature of the barriers to fungal invasion of the root interior in the Nod- mutants. We suppose that structural barriers or deficient signals halted the process of infection. Markwei and LaRue [7] observed no structural modification of the epidermis when the Myc- mutant roots were treated with Rhizobium, but noted epidermal thickening after they were exposed to pathogenic Pythium spp. We also did not see modification of the epidermis in either the mutants or the controls after contact with G. margarita. Thus, if epidermal thickening is a defense reaction, it does not explain the resistance of the mutants to VAM invasion. Since these mutants do not permit penetration by Rhizobium, showing no root hair curling, it would seem likely that they fail to perceive and react to signals necessary to the interaction. Until now, Myc- genotypes have been detected in symbiotic mutants of three commercially important legume species, faba beans, peas [5] and alfalfa [6]. The correlation between the Nod- and Myc- phenotypes raises the possibility that VAM fungi rely on the same signals as Rhizobium to initiate a favorable reaction from the plant. There is no fossil record to indicate when nodulation and mycorrhization evolved, but we
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can speculate that the use of a chitin-like glucosamine nod factors by Rhizobium and VAM fungi was derived from an ancestral plant/ microorganism interaction [29]. This hypothesis can be pursued by attempting to find homologues to nod genes in VAM fungi. The practical problem of culturing VAM fungi remains, but if useful information can be gained from Rhizobium [30] concerning the basic nature of the interaction, culture of VAM fungi may be facilitated. Furthermore, transformed roots can be cultured on a large scale, making it possible to collect large quantities of root exudate, which should aid in the search for substances produced by roots that promote the growth of VAM fungi.
Acknowledgments We wish to thank Dr Horst Vierheilig, Dr Preman Singh and Paul de la Bastide, Universit6 Laval for their critical reviews of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council (Canada) through a grant to Y. Pich6 and by the Cooperation France-Quebec to D. Tepfer and Y. Pich6. B. Balaji acknowledges CIES (Centre International des Etudiants et Stagiaires) for financial assistance during his stay in France.
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